A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi.pdf

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A manganese transporter, BB0219 (BmtA), is requiredfor virulence by the Lyme disease spirochete,Borrelia burgdorferi Zhiming Ouyanga, Ming Heb, Tara Omanb, X. Frank Yangb, and Michael V. Norgarda,1

aDepartment of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and bDepartment of Microbiology and Immunology,Indiana University School of Medicine, Indianapolis, IN 46202

Communicated by Jonathan W. Uhr, University of Texas Southwestern Medical Center, Dallas, TX, December 19, 2008 (received for reviewDecember 4, 2008)

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is

transmitted to mammalian hosts through an arthropod (tick) vec-

tor. To establish infection, Bb must acquire essential nutrients,

including transition metals, from its mammalian and tick hosts.

Thus far, no metal transporter has been identified in Bb. Here, we

report the identification of the first metal transporter, BmtA

(BB0219), in Bb. BmtA-deficientmutants of virulent Bb werereadily

generated, and the mutants grewslightlyslower thanwild-type Bb

in vitro. However, BmtA mutants were sensitive to the chelating

actions of EDTA, suggesting a role for BmtA in metal utilization.

Intracellular accumulation of manganese (Mn) was substantially

diminished in the bmtA mutant, indicating that BmtA was opera-

tive in Mn uptake. Given that BmtA lacks homology to any known

Mn transporter, we postulate that BmtA is part of a novel mech-

anism for Mn acquisition by a bacterial pathogen. BmtA also was

essential to the infectious life cycle of Bb in ticks and mammals,

thereby qualifying BmtA as a new borrelial virulence factor. In

addition, the bmtA mutant was sensitive to treatment with t -butyl

hydroperoxide, implying that BmtA, and thus Mn, is important to

Bb for detoxifying reactive oxygen species, including those poten-

tially liberated by immune effector cells during the innate immune

response. Our discovery of the first molecule involved in metal

transport in Bb provides a foundation for further elucidating metal

homeostasis in this importanthuman pathogen, which may lead to

new strategies for thwarting Lyme disease.

metal transport pathogenesis

L yme disease, caused by the spirochetal bacterium Borrelia burgdorferi ( Bb), remains the most common vector-borne

disease in the United States (1). Bb has a complex life cycleinvolving an arthropod ( Ixodes scapularis tick) vector and variousmammalian hosts (2, 3). In recent years, extensive efforts havebeen directed toward elucidating the mechanisms by which Bbcycles, adapts, and sustains itself in these diverse niches. It is now

well established that outer surface (lipo)protein C (OspC),OspA/B, decorin-binding protein B/A (DbpB/A), PncA, andBptA are required by Bb for efficient infection of ticks or

mammalian hosts (4–10). It is also generally accepted that therecently discovered Rrp2-RpoN ( 54)-RpoS ( S) regulatorypathway plays prominently in Bb’s vir ulence expression (11–14).

Transition metals, including iron (Fe), zinc (Zn), and man-ganese (Mn), are critical to both bacterial metabolism and

virulence (15–18). However, transition metals are tightly seques-tered within mammalian body fluids, making the scavenging of these cofactors by bacteria challenging. To overcome this, bac-teria have evolved a number of elegant systems for acquiringtransition metals from their environments. For example, Esch- erichia coli possesses multiple iron uptake systems (18, 19), theMntH Mn transporter (17, 20), and 2 Zn transporters (ZnuABCand ZupT) (16, 21). By tight regulation of these systems, bacteriamaintain metal homeostasis.

Compared w ith other bacterial pathogens, little is knownabout transition metal acquisition and homeostasis in Bb. Ge-nome sequence analysis has implied that Bb lacks genes encodingFe-acquisition systems (22), suggesting unconventionally that Femay not be required by this pathogen. This assertion wascorroborated by an elegant study showing that Bb accumulatesMn, rather than Fe, when grown in a serum-free medium (23).However, evidence has been lacking regarding whether Bbrequires Mn for growth and proliferation and, more importantly,to what extent Mn is needed by Bb to establish mammalian

infection and how Bb acquires this element.Blast analysis of the Bb genome has not revealed obvious

homologs of known Mn transporters. However, the Bb chromo-somal gene bb0219 encodes a GufA homolog with unknownfunction (22). Upon comprehensive sequence analysis, we dis-covered that BB0219 possesses the signature sequence (Axxx H-NxxxGLAVG) for the ZIP family [zinc-regulated metal trans-porters (ZRTs) and iron-regulated metal transporters(IRTs)-like protein family] (24), whose members typically are competentfor the uptake of Zn, Fe, or Mn (16, 21, 25, 26). Thus, wepostulated that bb0219 may be involved in the uptake of one ormore transition metals by Bb. To examine this possibility, wegenerated isogenic bb0219 mutants and c omplemented strains of

virulent Bb and examined their phenotypes with respect to metalaccumulation and their abilities to infect ticks and mice.

Results

In SilicoAnalysisof BB0219, a GufA Homolog. Blast searches revealedthat, in addition to Bb, functionally uncharacterized homologs of BB0219 exist in other bacteria, such as Borrelia garinii, Borrelia afzel ii, Clostridium botulinum, Methanococcus maripaludis, Desulfotalea psychrophila, Staphylococcus epidermidis, and Liste- ria monocytogenes. Originally, bb0219 was annotated as GufA, aprotein with unknown function (22, 27). GufA proteins havebeen placed as a subgroup of the larger ZIP family (25). Inbacteria, some ZIP proteins transport not only Zn2, but alsoother cations, such as Mn2, Fe2, and Cd2 (16, 21, 25). Allfunctionally characterized ZIP proteins contain a conservedtopology with 8 transmembrane domains. This topology also was

predicted to be in BB0219 when we analyzed BB0219 by usingprotein topology prediction programs PSORTb (28) and TM-HMM 2.0 (26). Most ZIP proteins contain a metal-binding site(HxHxH) between spanners III and IV (24), and all ZIP proteins

Author contributions: Z.O. and M.V.N. designed research; Z.O., M.H., T.O., and X.F.Y.

performedresearch; Z.O., M.H., T.O., and X.F.Y. analyzed data;and Z.O.and M.V.N. wrote

the paper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: michael.norgard@

utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/

0812999106/DCSupplemental .

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have a signature sequence (Axxx HNxxxGLAVG) in the mostconserved spanner IV (of which the conserved central histidineprovides an intramembranous heavy metal ion-binding site)(24). When we analyzed BB0219, we noted that although themetal-binding motif HxHxH was not apparent, the predictedprotein possesses the signature sequence (Axxx HNxxxGLAVG)for the ZIP family (24) in the putative spanner IV region. Thesein silico analyses thus implicated BB0219 in metal uptake by Bb.

bb0219 Is Cotranscribed Within a 6-Gene Operon. In the Bb genome, bb0219, bb0220, bb0221, bb0222, bb0223, and bb0224 are ori-ented in the same direction (Fig. 1 A). In this region, bb0219 isseparated from bb0220 by 40 bp, bb0223 is separated from bb0222 by 20 bp, and no intergenic regions are predictedbetween the other adjacent genes (22). To determine whetherthese genes are cotranscribed, RT-PCR was performed on RNA by using specific primers [supporting information (SI) Methodsand Table S1]. As shown in Fig. 1 B (lane 5), a fragment wasamplified by using the primer pair spanning the junction of

bb0219 and bb0220. Similar results were obtained for primerpairs spanning the junctions of bb0221 and bb0220 (lane 6), bb0222 and bb0221 (lane 7), bb0223 and bb0222 (lane 8), and bb0224 and bb0223 (lane 9), but not in the amplification usingprimers spanning bb0225 and bb0224 (lane 10). Results from allpositive and negative controls were as expected (Fig. 1 B, lanes2–4). These data indicatethat bb0219 –bb0224 are cotranscribed.

Inactivation and Complementation of bb0219 in Bb . bb0219 disrup-tion mutants were constructed via homologous recombination(Fig. 2 A). Through allelic exchange, a 740-bp internal fragmentof the 822-bp ORF of bb0219 was replaced with the P flgB-kancassette, yielding 2 kanamycin-resistant transformants,OY04/D4 and OY04/F42. To complement the bb0219 mutants,the shuttle vector pOY15 was created by fusing bb0219 to the Bb

flaB promoter P flaB (Fig. 2 A). After electroporation of pOY15into OY04/D4 or OY04/F42, 2 corresponding complementedclones, OY06/D11 and OY06/F6, were created. In addition, 2mock-complemented strains, OY07/F1 and OY07/F62, also weregenerated by transforming the empty shuttle vector pJD54 intoOY04/D4 or OY04/F42, respectively. The inactivation andcomplementation of bb0219 in these strains were confirmed byusing PCR amplification (Fig. 2 B), and the insertion and orien-tation of the P flgB-kan cassette within the disrupted bb0219 gene

were confir med by sequence analysis. Complementation also was confir med by recover y of the shuttle vector from thecomplemented strains.

To assess the expression of bb0219 in Bb strains, RT-PCRemploying specific primers was performed to detect transcripts.

As expected, bb0219 transcripts were detected in both WT 297and the complemented strains, but not in the mutants and themock-complemented clones (Fig. 2C). Borrelia plasmids, espe-cially those that are virulence-associated (such as lp25 andlp28–1), are easily lost during in vitro genetic manipulation,culminating in a loss of borrelial virulence (29). Hence, PCR-based plasmid profiling was performed to ensure that all plas-mids were retained in the mutant and complemented strains. Asshown in Fig. S1, the bb0219 mutant OY04/D4 contained thesame plasmid profile as that of WT 297. In addition, the bb0219mutant clone OY04/F42, the c omplemented strains (OY06/D11

and OY06/F6), and the mock-complemented strains (OY07/F1and OY07/F62) also contained all plasmids present in WT 297.

Involvement of BB0219 in Metal Uptake. Under darkfield micros-copy, the bb0219 mutant exhibited spirochetal morphologyidentical to that of the 297 parental strain. Mutant spirochetesgrew slightly slower than wild t ype in Barbour-Stoenner-Kelly II(BSK-II) medium (approximately a 3.6-fold difference in spiro-chete numbers between the WT and mutant after 9 days; Fig.S2). When the bb0219 mutation was complemented by usingpOY15, spirochetal growth was restored to the same level as W T297. To determine whether the mutant had a defect in growth ina mammalian environment, borreliae were cultivated in dialysismembrane chambers (DMCs) implanted into the peritoneal

Fig. 1. bb0219 is cotranscribed with bb0220– bb0224. ( A) Schematic repre-

sentation of the putative bb0219– bb0224 operon in Bb. (B) Results from

RT-PCR. Lane 1, molecular weight markers; lane 2, primer pair a1 and a2 in

ordinary PCRusing genomic DNA as template;lane3, primerpaira1 anda2 in

ordinaryPCR usingRNA astemplate(noRT control);lane4, primerpaira1 and

a2; lane 5, primer pair a3 and b1; lane 6, primer pair b2 and c1; lane 7, primer

pairc2 and d1; lane8, primerpair d2ande1;lane 9,primer paire2 and f1; and

lane 10, primer pair f2 and g1.

Fig. 2. Construction of bb0219 disruption mutants and complemented

strains. ( A) Schematic representation of bb0217–bb0220 genes in the Bb

chromosome, insertionof the PflgB-kangene cassette by homologous recom-

bination, and the relevant complementation plasmid. Arrows indicate the

approximate positions of the oligonucleotide primers used for subsequent

PCR analyses. (B) PCR analysis of WT 297, bb0219 mutants, and the comple-

mented strains. The bb0219-specific primerpairs used in PCRare indicatedon

the right. Lane 1, WT 297; lane 2, bb0219 mutant OY04/D4; lane 3, mutant

OY04/F42; lane 4, complemented strain OY06/D11; lane 5, complemented

strain OY06/F6; lane 6, mock-complemented strain OY07/F1; and lane 7,

mock-complemented OY07/F62. (C ) RT-PCR was used to determine the pres-

ence or absence of bb0219 transcripts. Lanesand primer pairdesignationsareas in B.

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cavities of rats. After 12 days in DMCs, spirochetes of Bb strains297, the bb0219 mutant, and the complemented strain increasedcomparably from 103 /mL to 107 /mL. These combined findingssuggest that bb0219 is not essential for Bb growth either in vitroor in a surrogate mammalian model system.

Because bb0219 was postulated to encode a metal transporter, we first examined the response of Bb to metal limitation bycomparing the growth of WT 297, bb0219 mutants, and com-plemented strains under metal-restricted conditions. As shownin Fig. 3 A, the growth of the bb0219 mutants (OY04/D4 andOY04/F42) was inhibited when bacteria were cultivated in thepresence of EDTA. Growth of the bb0219 mutants was com-pletely inhibited in the presence of 0.1 mM EDTA, whereas WT297 and the complemented strains (OY06/F6 and OY06/D11)grew well under this condition. This metal-associated phenotypeimplies that BB0219 is related to metal acquisition by Bb,prompting us to rename bb0219 as bmtA for Borrelia metaltransport protein A.

To garner more direct evidence for the involvement of BmtA in metal transport, we measured the intracellular metal contentof various Bb strains. We hypothesized that if BmtA indeed is ametal transporter, the accumulation of one or more metals thatBmtA transports would be impaired in BmtA-deficient mutants.To assess this, borreliae grown in BSK-II medium were har-

vested and lysed, and the metal contents within the hydrolysates were determined. We found 174 or 81 ng of Mn in 2.5 1010

spirochetes of the WT or complemented strain, respectively (Fig.

3 B). In contrast, Mn was not detected (below the detection limitof 10 ng) in the same numbers of spirochetes of the bmtA mutantor the mock-complemented strain. Because in silico analysesindicated that BmtA might be a Zn-transporting ZIP protein, wealso compared the Zn contents among these isolates. Contraryto our expectations, intracellular Zn accumulation was notreduced in the BmtA-deficient mutants (Fig. 3C). In fact, theBmtA-deficient mutants appeared to accumulate more Zn thanthe WT or the complemented strains (Fig. 3C). The BmtA-deficient mutants also accumulated magnesium (Mg) at levelscomparable to those of WT 297. The combined data indicatethat bmtA encodes a protein involved in Mn transport, ratherthan in Zn or Mg transport, in Bb.

We also investigated Bb’s sensitivity to Mn as a surrogatemarker of Mn uptake. When spirochetes were grown in BSK-II

medium containing exogenously added Mn, WT 297 was sub-stantially more sensitive to Mn toxicity (60% growth inhibitionat 50 M Mn) than the BmtA-deficient mutant (no growthinhibition at 50 M Mn), as we predicted (Fig. S3). These data

were consistent with the conclusion that BmtA is involved in theuptake of Mn by Bb.

BmtA Is Essential for Bb to Establish Mammalian Infection. Toinvestigate the contribution of bmtA to Bb mammalian infectiv-ity, C3H/HeN mice were infected intradermally with various Bbstrains. After 4 weeks, skin, heart, and joint tissues were col-lected and transferred into f resh BSK-II medium. Cultures weremonitored for 4 weeks for spirochete growth. Whereas motilespirochetes were recovered from tissues of all mice inoculated

with 104 spirochetes of either the WT or complemented strains

(OY06/F6 and OY06/D11), no bacterial growth was obser ved inany cultures of tissues from mice infected with either 104 or 107

of the bmtA mutants (OY04/D4 and OY04/F42) or mock-complemented strains (OY07/F1 and OY07/F62) (Table 1).These results demonstrate that bmtA is essential for mammalianinfectivity by Bb.

BmtA Is Required by Bb to Infect Ticks. Because the bmtA mutant was unable to infect mice, it was not possible to examine thecontribution of BmtA to the entire Bb infectious cycle betweenticks and mammals (mice). Therefore, as an alternative means toassess the ability of the bmtA mutant to infect, multiply, andsurvive in ticks, we used a microinjection technique to introducespirochetes into sterile nymphs. Microinjected ticks carrying

various Bb strains then were allowed to feed on naïve mice, and

naturally detached ticks were collected and dissected, and ticktissue contents were subjected to immunofluorescence assays.

As shown in Fig. 4, in contrast to W T 297 and the complementedstrain (OY06), the bmtA mutant (OY04) was unable to colonizeand survive in ticks. Comparable results also were obtained whenlarval ticks were alternatively infected by using a tick immersionmethod (30). The combined data demonstrated that bmtA isrequired by Bb to infect and survive in its arthropod vector.

BmtA Is Important for Protecting Bb Against Reactive Oxygen Species

(ROS). In many bacteria, Fe is involved in antioxidant activities,primarily through the ability of Fe2 to reduce H2O2 and byfunctioning as a key cofactor of superoxide dismutase (SodA)(15). Given the facts that (i) Bb does not accumulate iron (23)

Fig. 3. Role of metals in spirochete growth. In all experiments, BSK-II

medium was inoculated with 1,000 spirochetes per mL. After growth at 37 °C

for 9 days, spirochetes were collected and enumerated by darkfield micros-

copy. Datawere derived from3 independent experiments. Error bars indicate

standard deviations, and the asterisk indicates statistical significance using

Student’s t test (P 0.05). ( A) Metal ions were depleted from BSK-II medium

by adding varying amounts of EDTA; bacterialgrowth under these conditions

was monitored. (B and C ) Spirochetes from 200 mL of culture were collected,

andtheintracellular Mn (B)a n d Z n (C ) contentswere measuredby inductively

coupled plasma atomic emission spectrometry. Values are shown as nano-

grams per 2.5 1010 spirochetes of strains WT 297, bb0219 mutant OY04/D4,

complementedstrainOY06/D11, or mock-complemented strain OY07/F62. For

Mn contents in OY04 and OY07, the detection limit (10 ng per 2.5 1010

spirochetes) is represented, as values were below this threshold.

Ouyang et al . PNAS March 3, 2009 vol. 106 no. 9 3451







but does accumulate Mn (Mn accumulation was severely im-paired in the bmtA mutant), (ii) Mn is believed to be involved inthe oxidative stress response of a number of pathogens (17, 31,32), and (iii) Bb SodA (BB0153) has been predicted to beMn-dependent (22, 33), we explored whether the inactivation of bmtA adversely impacted Bb’s ability to detoxify intracellularsuperoxide. When WT 297 was exposed to 5 mM or 10 mMt-butyl hydroperoxide, 100% or 93%, respectively, of the cellssurvived (Fig. 5). In contrast, the bmtA mutant was significantlymore sensitive to t-butyl hydroperoxide, with a survival of 50%at 5 mM t-butyl hydroperoxide. These data suggest that BmtA and Mn play important roles in the detoxification of ROS (i.e.,oxidative stress response).

Discussion

Analyses of genomic sequence information have suggested thatthe utilization of transition metals by Bb may be dramatically

different from other bacteria. For example, in contrast to mostbacterial pathogens, Bb does not appear to require iron tosupport its growth (23). Rather, it was reported that Bb accu-mulated Mn when grown in a serum-free medium (23), but the

relevance of this finding to what occurs when Bb is cultivated inmore nutrient-rich (e.g., serum-containing) environments, or what Bb encounters in ticks or mammalian hosts, has remainedobscure. It additionally has been unknown to what extent Mnmay be important for establishing mammalian infection by Bb.

Employing molecular genetics to create BmtA mutants andcomplemented strains, here we have identified what appears tobe a novel Mn transporter in Bb. We derived this conclusionfrom 2 key observations. First, BmtA has a predicted topologysimilar to Zn transporters (ZIP proteins) (25); in our analyses,BmtA was predicted to be a cytoplasmic membrane proteincontaining 8 transmembrane domains (26, 28). Second, our dataclearly showed that BmtA is critical for Mn uptake in Bb, at least

when Bb is grown in BSK medium. Therefore, it is likely that bmtA encodes a transporter mediating the uptake of Mn across

the cytoplasmic membrane of Bb.Our study is significant in at least two major ways. First, to our

knowledge, no metal transporter had thus far been reported inthis important human pathogen. Second, 3 classes of Mn trans-porters have been characterized in prokaryotes, including thenatural resistance-associated macrophage protein (Nramp) typeMn transporter, the ATP-binding cassette (ABC) Mn permease,and the P-type ATPase Mn transporter (15, 17, 34). Sequence

Table 1. Infectivity of B. burgdorferi clones in mice

Strain, clone Description Dose

No. of cultures positive/total no. of

specimens examinedNo. of mice infected/

total no. of miceHeart Joint Skin All sites

297 WT B. burgdorferi 104 7/7 7/7 7/7 21/21 7/7

OY04/D4 297, bmtA* 104 0/6 0/6 0/6 0/18 0/6

OY04/F42 297, bmtA 104 0/6 0/6 0/6 0/18 0/6

OY04/D4 297, bmtA 107 0/5 0/5 0/5 0/15 0/5

OY04/F42 297, bmtA 107 0/5 0/5 0/5 0/15 0/5OY06/F6 OY04/D4 transformed with pOY15 104 3/3 3/3 3/3 9/9 3/3

OY06/D11 OY04/F42 trans formed with pOY15 104 3/3 3/3 3/3 9/9 3/3

OY07/F1 OY04/D4 transformed with pJD54 104 0/3 0/3 0/3 0/9 0/3

OY07/F62 OY04/F42 transformed with pJD54 104 0/3 0/3 0/3 0/9 0/3

OY07/F1 OY04/D4 transformed with pJD54 107 0/3 0/3 0/3 0/9 0/3

OY07/F62 OY04/F42 transformed with pJD54 107 0/3 0/3 0/3 0/9 0/3

*bmtA: bmtA mutant.

Fig.4. BmtAis requiredby Bb to infect andsurvivein ticks. Unfed I. scapularis

nymphs were microinjected with various Bb strains and were allowed to feed

to repletion on normal mice. After detachment, fed nymphs were dissected,

and the tick tissue contents were subjected to immunofluorescence assays

with FITC-labeledanti-Bb antibody.( A) Confocalimmunofluorescencemicros-

copy; spirochete morphology is evident from tissues of ticks infected with WT

297or thecomplemented (OY06) strains.(B) Quantitativeassessment (number

of borreliae per microscope field) of the data shown in A; WT 297 was

considered the 100% value.

Fig. 5. The bmtA mutant is sensitive to treatment with t -butyl hydroperox-

ide. Bb (WT 297 or bmtA mutant OY04/D4) was grown to a cell density of 1

107 cells/mLand thentreated withvarying amounts of t -butyl hydroperoxide.

Averages with standard deviations are shown. The asterisk denotes a signif-

icant difference in t -butyl hydroperoxide sensitivity (P 0.05).




similarity searching revealed that BmtA has no homology to anyof these bacterial Mn transporters. Despite this, we found thatthe ZIP domain-carrying BmtA protein is involved in thetransport of Mn but not Zn. This is particularly striking, giventhat ZIP domains typically are involved in Zn transport (25).Our study thus not only provides further insights into thephysiological functions of bacterial ZIP proteins, but also sug-gests that BmtA and its homologs comprise a novel mecha-nism(s) for Mn acquisition.

Because of their similar ionic radius and chelate structures,Mn2, Fe2, Mg2, and Zn2 are interchangeable in the metal-binding site of many proteins. In this regard, Mn in place of ironhas been proposed to be required by Bb (23). Unlike mostpathogens that possess overlapping or redundant Mn transport-ers, Bb probably expresses only one Mn transporter, BmtA, atleast when borreliae are grown in vitro. This contention isstrongly supported by our data that Mn was not detected in theBmtA mutant. Regarding the fact that the mutant had nosubstantial growth defect in vitro or in DMCs, we propose thatMn is not an obligate requirement for Bb physiology, althoughit may facilitate optimal growth of borreliae in v itro. Because Znmay substitute for activities typically served by Fe or Mn, whenMn is not available in the environment, Bb may switch itsrequirement from Mn to Zn. Consistent with this assertion, our

data showed that the bmtA mutant accumulated more Zn thanthe WT strain, ostensibly through the activation of one or morepotential Zn transporter(s). Transcriptional profiling compari-sons between WT, bmtA mutants, and complemented strainsmight shed added light on this possibility. Alternatively, inaddition to the import of Mn, BmtA may also be involved in theefflux of Zn, in so much that this dual import–export functionhas been noted for other bacterial metal transporters (e.g.,CorA) (35). Finally, it remains possible that the physiologicalfunctions of Mn also may be served by Mg because of theirsimilar structure.

Inactivation of bmtA rendered Bb completely noninfectiousfor mice and unable to infect and proliferate in ticks, indicatingthat BmtA is essential for Bb’s mammalian infectivity and

colonization of its arthropod vector, even though BmtA and Mnare dispensable when Bb is cultivated in vitro. The contributionof Mn transporters and Mn to bacterial pathogenesis has been

well established in many other pathogens, including in those where Mn is not an obligate requirement for bacterial growth in vitro (15, 17, 31). Mn typically is not a widely used cofactor inbiology and serves as a physiological cofactor for only a handfulof enzymes (15, 17). Therefore, if Mn is dispensable whenbacteria are cultivated in vitro, why is it important for bacterial

virulence? In the case of Bb, one possibility is that BSK mediumdoes not recapitulate the physiological or metal conditions that Bb encounters in either ticks or mammals. However, there are anumber of additional observations in our study that may addressthis question. First, in c omparison with WT, the Bb bmtA mutant

was sensitive to t-butyl hydroperoxide, suggesting that BmtA

(and thus Mn) is probably involved in the detoxification of ROS. Akin to the situation for other bacterial pathogens (17, 31, 32),the most obvious role for BmtA and Mn likely is in the Bboxidative stress response. For example, in the process of tickfeeding or during the process of mammalian host dissemination,BmtA and Mn may be required by Bb to counter superoxidecontained in mammalian blood (36, 37). Further, BmtA and Mnmay also play a role in defending the spirochete from the initialhost immune response, including the extremely deleteriouseffects of ROS generated during the innate host immune re-sponse to Lyme borreliosis (36). Although entirely speculative,borrelial Mn-dependent SodA also may participate in one ormore of these events. Finally, in a wide variety of bacterialpathogens, Mn contributes to virulence by regulating the ex-

pression of many virulence determinants (15, 17, 31). Similarly,Mn, possibly via the Mn-dependent Fur homolog BosR (23, 33,37, 38), might play a critical role in controlling Bb virulence.Future microarray studies may shed additional light on to whatextent BmtA, and by inference Mn, regulates virulence expres-sion in Bb.

Our study has demonstrated that the Lyme disease spirocheterequires BmtA’s Mn-associated transport function(s) to main-tain its infectious cycle in nature. To date, only relatively few

virulence factors, including OspC, DbpB/A, OspA/B, PncA, andBptA, have been identified in Bb, and the functions of most of these proteins remain obscure (4, 5, 7–10). In this regard, ouridentification of BmtA as a novel vir ulence factor in Bb providesfurther insights into molecular mechanisms that contribute to Bb’s survival in nature and may lead to new strategies tointerrupt the spirochete life cycle. In addition, our findingsrepresent a first step in elucidating transition metal homeostasisin Bb. Our study also prompts a number of salient questions.First, considering the predicted topology of BmtA, we proposethat the theoretical 8 transmembrane segments assemble achannel to allow Mn or other ions to pass through the cytoplas-mic membrane. However, how the channel is opened or closed,how the metal ions are transported, or whether energy isrequired remain completely unknown. It also is unknown

whether BmtA is involved in the transport of other cations, as theaffinities and selectivity of BmtA for Mn and other cations havenot yet been fully assessed. Biochemical experiments with re-combinant BmtA and, possibly, reconstitution into liposomesmay be strategic for addressing some of these questions. Second,

what are the functional relationships between the other 5members of the bmtA operon? Although none of these 5 proteins

was predicted to be implicated in metal transport, it remainspossible that they indeed contribute to Mn transport. Inactiva-tion of each gene of the operon may shed additional light on thisissue. Finally, what are the specific cellular processes in Bb for

which Mn is required? In many pathogens, Mn transport iscontrolled by an Mn-specific transcription factor, MntR, as wellas by Fur, PerR, or Ox yR (15, 17, 31). Although BosR in Bb was

predicted to be Mn-dependent (23), it is not clear whether bmtAis regulated by BosR or responses to the intracellular Mn pool.Examination of bmtA transcriptional control using reporterassays may help to clarify the regulation of bmtA.

MethodsBacterial Strains and Culture Conditions. Infectious Bb strain 297 (39) was used

as the WT strain throughout this study. Bb was cultured at 37 °C in BSK-II

medium (40) supplemented with 6% rabbit serum (Pel-Freeze). To assess the

growth of Bb in a mammalian host-adapted state, borreliae were also culti-

vated within DMCsimplanted intothe peritoneal cavitiesof rats, as previously

described (41). All animal experiments were approved by the Institu-

tional Animal Care and Use Committee at UT Southwestern Medical Center

(Dallas, TX).

Construction of bmtA Mutants and Complemented Strains. The bmtA mutant

strain OY04 was created by allelic exchange in Bb 297 using a suicide vectorpOY04. The mutation in bmtA was complemented by transforming a shuttle

vector, pOY15, based on the pJD54 plasmid, into the bmtA mutant, generat-

ing OY06. In addition,a mock-complemented mutant, OY07, was constructed

by transforming pJD54 into Bb OY04. All plasmid constructs were character-

ized by PCR, restriction digestion, and sequence analysis (SI Methods). Trans-

formation of Bb was performed as previously described (12, 14). Plasmid

contents of all Bb strains were determined by PCR using specific primers (4).

Intracellular Metal Contents. Intracellular metal contents of borreliae were

measured as previously described (19). Briefly, bacterial cultures grown to

stationary phase were harvested, and the pellets were lysed by using 65%

HNO3 (TraceMetal grade; Sigma). The metal contents in the lysates were

measured by using inductively coupled plasma atomic emission spectrometry

by the Research Analytical Laboratory, University of Minnesota (St. Paul, MN;

Ouyang et al . PNAS March 3, 2009 vol. 106 no. 9 3453






SI Methods). Three independent tests were performed, and the results were

analyzed by using Student’s t test.

Sensitivity of Bb to ROS. This test was performed as previously described, with

modifications (36, 37). Briefly, when spirochete growth reached a cell density

of 5 107 cells/mL, the culture was divided into 5 aliquots and treated with

varying concentrations of oxidants (0 –20 mM t -butyl hydroperoxide) at 37 °C

for 4 h. After incubation, cells were serially diluted in fresh BSK medium in a

96-well plate and incubated for 2 weeks. Percent survivability was calculated

as thenumber of clonesrecovered from thetreatedgroup versusthe number

of clones recovered from the untreated group.

Bb Infection of Mice. The infectivity of Bb clones was assessed by using the

murine needle-challenge model of Lyme borreliosis (42). C3H/HeN mice

(Charles River Laboratories) were infected via intradermal injection with

various concentrations of Bb. At 4 weeks after inoculation, skin, heart, and

joint tissues were collected and cultured in BSK supplemented with 1 Bor-

relia antibiotic mixture (Sigma). The outgrowth of spirochetes in these cul-

tures was assessed by using darkfield microscopy.

Artificial Infection of Ticks with Bb . I. scapularis nymphs or larvae (obtained

from the Tick Rearing Facility at Oklahoma State University, Stillwater, OK)

were artificially infected with various strains of Bb by using a microinjection

technique (11, 43) or an immersion method (30) (see SI Methods). After

infection, tickswere fedto repletionon separatenaïve C3H/HeN mice. Subsets

of ticks were dissected immediately and analyzed by immunofluoresence

assay (SI Methods). Spirochetes were stained with the BacTrace FITC-

conjugated goat anti-Bb antibody (Kirkegaard and Perry Laboratories) andobserved by using an Olympus BX50 fluorescence microscope.

ACKNOWLEDGMENTS. We thank Farol Tomson and Eric Hansen for criticalreading of the manuscript. This work was supported by National Institutes ofHealth Public Health Service Grant AI-059062 from the National Institute ofAllergy and Infectious Diseases.

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A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi.pdf